New approaches to lower intraocular pressure, a significant risk factor for the progression of glaucoma, will be investigated. Outflow through the trabecular meshwork will potentially be enhanced by ILK, PI3K and Src kinase inhibitors, which have been shown to alter the cytoskeleton and cellular adhesions of trabecular meshwork (TM) cells in culture. The effects of these compounds on intraocular pressure (IOP) and outflow facility will be studied in organ-cultured anterior segments and in monkey eyes in vivo. Gene therapy approaches will be utilized to lower IOP by enhancing outflow via the uveoscleral pathway. One of the current most effective pharmacotherapies for glaucoma is based on derivatives of prostaglandin (PG)F2a which binds to the FP receptor leading, in part, to enhanced matrix metalloproteinase (MMP) synthesis. This, in turn, alters the composition of the extracellular environment of the ciliary muscle (CM) and sclera leading to an enhancement of uveoscleral outflow. Lentiviral vectors carrying genes for PGF synthase and for stromelysin (MMP-3) will be injected into the anterior segment of monkey eyes in vivo and the effects on IOP and uveoscleral outflow will be assessed. PGF2a and MMP-3 production will also be assessed in the aqueous humor and in the media from human TM and CM cells transduced in vitro. A final objective will be to investigate in vivo the role of the neurotransmitter, nitric oxide, in regulating IOP, aqueous humor inflow and outflow. To simulate the dramatically reduced levels of nitric oxide synthase (NOS) in the anterior segment in glaucoma, a short-term equivalent of nitrergic denervation will be created in monkeys in vivo following topical or intracameral dosing with the non-selective NOS inhibitor L-NAME and/or the relatively selective neuronal nitric oxide synthase inhibitors 7-NI and/or NPLA. The effects on basal pupil diameter, refraction, aqueous humor formation and/or outflow facility will be determined. Conversely, these parameters will be measured in response to the nitric oxide donor, SNP, in the presence and absence of cholinergic and/or adrenergic blockade. These results will provide important insights for developing new therapies to lower IOP. Studies will be conducted in living nonhuman primates, in vitro in organ-cultured anterior segments and in trabecular meshwork cells and ciliary muscle cells in culture. The following techniques will be employed: aqueous humor formation by fluorophotometry;outflow facility by two-level constant pressure perfusion;uveoscleral outflow calculated from isotope dilution and accumulation measures;IOP by Goldmann applanation tonometry;refraction by Hartinger coincidence refractometry;pupil diameter via vernier calipers;gene expression in vivo by coexpression of GFP fluorescence detected by a custom designed research microscope system;gene product detection by Western blots and enzyme immunoassays and immunohistochemistry. PUBLIC HEALTH RELEVANCE: The proposed studies will investigate new approaches to lower intraocular pressure that may be further developed for glaucoma therapy. The first two objectives are to enhance aqueous humor outflow through the two known outflow pathways using novel small molecules (PI3K, ILK and Src kinase inhibitors) to enhance trabecular outflow, and gene therapy (lentiviral vector mediated delivery of genes that increase production of enzymes (prostaglandin synthase and stromelysin)) to increase uveoscleral outflow. A third goal is to better understand the role of an important neurotransmitter (nitric oxide) in regulating normal and drug induced changes in aqueous humor formation and drainage in order to provide the basis for novel glaucoma therapies.